Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.     

IL-2 gene expression and NF-kappa B activation through CD28 requires reactive oxygen production by 5-lipoxygenase.

Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of interleukin-2 (IL-2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28-mediated activation of the NF-kappa B/CD28-responsive complex and IL-2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5-lipoxygenase. Our data suggest that lipoxygenase metabolites activate ROI formation which then induce IL-2 expression via NF-kappa B activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway.     

TNF-alpha and H2O2 induce IL-18 and IL-18R beta expression in cardiomyocytes via NF-kappa B activation

Myocardial ischemia/reperfusion is characterized by oxidative stress and induction of proinflammatory cytokines. Interleukin (IL)-18, a member of the IL-1 family, acts as a proinflammatory cytokine, and is induced during various immune and inflammatory disorders. Therefore, in the present study we investigated whether IL-18 expression is regulated by cytokines and oxidative stress in cardiomyocytes. TNF-alpha induced rapid and sustained activation of NF-kappaB whereas H(2)O(2) induced delayed and transient activation. Both TNF-alpha and H(2)O(2) induced IL-18 mRNA and precursor protein in cardiomyocytes, and IL-18 release into culture supernatants. However, only TNF-alpha led to sustained expression. Expression of IL-18Rbeta, but not alpha, was induced by both agonists. TNF-alpha and H(2)O(2) induced delayed expression of IL-18 BP. Pretreatment with PDTC attenuated TNF-alpha and H(2)O(2) induced IL-18 and IL-18Rbeta, but not basal expression of IL-18Ralpha. These results indicate that adult cardiomyocytes express IL-18 and its receptors, and proinflammatory cytokines and oxidative stress regulate their expression via activation of NF-kappaB. Presence of both ligand and receptors suggests IL-18 impacts myocardial biology through an autocrine pathway.     

T4 cell activation by immobilized phytohemagglutinin: differential capacity to induce IL-2 responsiveness and IL-2 production

Soluble mitogens, such as PHA induce accessory cell (AC)-dependent T cell proliferation. One function of the AC is to create a stimulatory matrix. Therefore, experiments were carried out to determine whether PHA immobilized onto microtiter plates could stimulate T cells in the absence of AC. Peripheral blood T4 cells were cultured under limiting dilution conditions with either soluble or immobilized PHA with or without rIL-1 beta, rIL-2, r-TNF-alpha, an anti-CD28 mAb (9.3), or irradiated EBV-transformed B cells as AC. The frequency of proliferating T4 cells was assessed by examining wells microscopically, and the frequency of T4 cells producing IL-2 was assessed by examining the ability of supernatants to support CTLL-2 proliferation. The percentage of T4 cells growing and producing IL-2 was determined by a maximum likelihood procedure. Immobilized, but not soluble, PHA induced a mean of 20.0 +/- 2.6% of T4 cells to grow in the complete absence of AC in medium supplemented with rIL-2. Whereas rIL-1 beta, rTNF-alpha, and 9.3 were unable to support T4 cell growth in the absence of rIL-2, each enhanced the percentage of T4 cells responding to immobilized PHA in the presence of rIL-2. In contrast, both soluble and immobilized PHA were unable to induce T4 cell IL-2 production in the absence of AC, even when cultures were supplemented with rIL-1 beta or 9.3. In the presence of AC, a small percentage of T4 cells (5.4 to 11.7%) was stimulated to produce detectable amounts of IL-2 by either immobilized or soluble PHA. Moreover, in the presence of AC, a very small population (approximately 1%) of PHA-stimulated T4 cells proliferated without supplemental rIL-2. The data indicate that a matrix of immobilized PHA is sufficient for some T4 cells to be activated to respond to IL-2, whereas others require additional signals provided by rIL-1 beta, rTNF alpha, 9.3, or AC. In contrast, neither soluble nor immobilized PHA is sufficient to induce T cell IL-2 production. This response requires signals provided by intact AC.     

Isolated peripheral T cells from GvHR recipients exhibit defective IL-2R expression, IL-2 production, and proliferation in response to activation stimuli

Graft-vs-host reactions (GvHR) following the injection of class I/II MHC disparate parental cells into unirradiated F1 recipient mice result in the development of marked immune dysfunction. Following negative selection using adherence and antibody and complement depletion, highly purified T cells were examined to determine their ability to undergo activation. Three weeks after GvHR initiation, unstimulated splenic T cells from GvHR mice displayed normal CD3 and IL-2R expression but elevated expression of class I MHC and Ly-6A/E antigens. Despite culture with normal F1 accessory cells, both CD4+ and CD8+ GvHR T cells exhibited low levels of proliferation to both Con A and anti-CD3 mAb. Although following exposure for 12 h to either of these stimuli, GvHR T cells expressed normal levels of IL-2R, expression was greatly decreased vs normal T cells between 24 and 48 h. In addition, at no timepoint was detectable IL-2 produced by GvHR T cells. Importantly, mixing experiments did not demonstrate detectable suppressive activity in the purified GvHR T cell subsets. GvHR T cells were also tested for their ability to respond to stimuli in the absence of any accessory cell population. These cells again did not proliferate to levels equivalent to normal T cells. Incubation with PMA and either cytokines (Con A supernatant, rIL-7) or anti-CD3 mAb resulted in only low levels of proliferation in GvHR T cells. Notably, at high ionomycin concentrations together with PMA, GvHR T cells did proliferate to equivalent levels as normal cells. However, with decreasing concentrations of ionophore, these cells failed to proliferate as well as normal cells. In total, these findings demonstrate that GvHR T cells are phenotypically and functionally distinct from normal T cells. The results suggest that GvHR T cells themselves may contribute to the well-characterized immune depression occurring in recipients undergoing GvHR.     

TLR3 activation evokes IL-6 secretion, autocrine regulation of Stat3 signaling and TLR2 expression in human bronchial epithelial cells

Human bronchial epithelial cells exposed to synthetic double-stranded RNA (poly I:C) exhibited increased IL-6 and RANTES secretion and TLR2 expression that was inhibited following TLR3 silencing. Increased NF-κB and Stat3 phosphorylation were detected after poly I:C exposure and pretreatment with neutralizing antibody targeting IL-6 receptor α (IL-6Rα -nAb) or blocking Jak2 and Stat3 activity inhibited Stat3 phosphorylation. TLR2 up-regulation by poly I:C was also reduced by IL-6Rα-nAb and inhibitors of Jak2, Stat3 and NF-κB phosphorylation, whereas RANTES secretion was unaffected, but abolished following NF-κB inhibition. Treatment with exogenous IL-6 failed to increase TLR2. These findings demonstrate that TLR3 activation differentially regulates TLR expression through autocrine signaling involving IL-6 secretion, IL-6Rα activation and subsequent phosphorylation of Stat3. The results also indicate that NF-κB and Stat3 are required for TLR3-dependent up-regulation of TLR2 and that its delayed expression was due to a requirement for IL-6-dependent Stat3 activation.     

Differential regulation of surface Ig- and Lyb2-mediated B cell activation by cyclic AMP. I. Evidence for alternative regulation of signaling through two different receptors linked to phosphatidylinositol hydrolysis in murine B cells.

The differential effect of cAMP on the regulation of early biochemical and cellular functions mediated through two different receptors on murine B cells are reported here. Surface IgM, the Ag receptor, and Lyb2, a 45-kDa differentiation Ag are concomitantly expressed on mature murine B lymphocytes. Triggering of B cells through these molecules, independently, resulted in inositol 1,4,5-triphosphate (IP3) generation, increase in intracellular Ca2+ levels, and cell enlargement associated with progression of cells from G0 to G1 ultimately resulting in DNA synthesis. Pretreatment of resting B cells with cholera toxin as well as other agents that raise the intracellular cAMP [(cAMP)i] such as forskolin, N6,2'-O-dibutyryl cyclic AMP, and 3-isobutyl-1 methyl xanthine inhibited the Ag receptor but not Lyb2-mediated DNA synthesis. The elevation of (cAMP)i inhibited the surface IgM but not Lyb2-mediated IP3 generation, Ca2+ response, and progression from G0 to G1 phase of the cell cycle. Failure of forskolin or N6,2'-O-dibutyryl cyclic AMP to inhibit Lyb2-mediated responses did not appear to be due to induction of cAMP-specific phosphodiesterase activity. Concentrations of H8 [N-(2-(methylamino)-ethyl)-5-isoquinoline sulfonamide, diHCl] inhibitory to cAMP dependent PKA prevented the inhibitory effect of forskolin on surface IgM-mediated Ca2+ response, suggesting that cAMP exerted its effects through PKA. These findings suggest that distinct PLC-coupled receptors, such as sIgM and Lyb2 molecules in B cells, may use either alternative mechanisms for phosphatidylinositol 4,5-bisphosphate hydrolysis or may use different intermediary transducer molecules that differ in their sensitivity to increased (cAMP)i levels. Thus "cross-talk" among cAMP and phosphatidylinositol signaling pathways was demonstrated for IgM but not Lyb2-mediated B cell activation.     

Molecular events in B cell activation. I. Signals required to stimulate G0 to G1 transition of resting B lymphocytes

Anti-immunoglobulin antibodies (anti-Ig) can stimulate a majority of resting B cells via their receptor Ig. Evidence suggests that the signals generated after this ligand-receptor interaction may be transduced via hydrolysis of inositol phospholipids. In other systems, the ability of inositol phospholipid hydrolysis to link receptor-ligand interactions to subsequent activational events has been suggested to relate to the ability of metabolic intermediates of this hydrolytic process to facilitate activation of protein kinase C and mobilization of Ca+2. In this study, we investigated the importance of protein kinase C and Ca+2 mobilization in the signaling mechanism by which anti-Ig drives B cells to undergo G0 to G1 transition. Our results show that pharmacologic inhibition of either protein kinase C activity or channel-mediated Ca+2 influx completely abrogates the increase in RNA synthesis associated with B cell activation after stimulation by anti-Ig. This suggests that pathways leading to both protein kinase C activation and elevation of intracellular Ca+2 are critical for receptor Ig-mediated G0 to G1 transition. Furthermore, studies in which anti-Ig-induced signaling could be bypassed by directly facilitating Ca+2 mobilization and protein kinase C activation using Ca+2 ionophore and phorbol diester show that these events are sufficient to drive the majority of resting B cells into G1 in the absence of additional signaling from accessory cells or extra-cellular factors. However, like anti-Ig-induced stimulation, Ca+2 ionophore and phorbol diester are relatively inefficient in driving B cells that have entered G1 into S phase. We discuss the relevance of these results towards the transduction mechanism linking B cell membrane-associated Ig-generated signals with subsequent activation events.     

Polyclonal B cell activation by B cell differentiation factor B151-TRF2. I. Involvement of self-Ia recognition process mediated by B cells

The present study examined the functional role of Ia antigens on B cells in polyclonal B cell activation induced by a B cell differentiation factor, B151-TRF2. The polyclonal IgM PFC responses by B151-TRF2 were inhibited by monoclonal antibodies specific for class II MHC antigens (Ia antigens) but not class I MHC antigens. Such inhibition by anti-Ia antibodies was haplotype-specific and was observed in the absence of both T cells and accessory cells. Moreover, the anti-Ia antibody-induced inhibition of the B151-TRF2 responses was not due to the blocking of binding of B151-TRF2 to the corresponding B cell receptor. A series of kinetic studies revealed that some Ia-mediated cellular activation process occurs before the resting B cells become responsive to B151-TRF2. Thus, the B151-TRF2-mediated B cell responses consist of at least two distinct phases. The early phase is an Ia-dependent but B151-TRF2-independent process, whereas the late phase is an Ia-independent but B151-TRF2-dependent process. To further characterize the functional role of Ia antigens on B cells, an additional experiment was carried out by using F1 B cells which co-dominantly express both parental Ia antigens on the surface. Interestingly, it was observed that the degree of inhibition of the B151-TRF2-mediated responses of F1 B cells by anti-parental Ia antibody was, at best, one-half that of the parental B cells, suggesting that F1 B cells may be separated into two subpopulations with the restriction specificity for the respective parental Ia antigens. To examine this possibility, (B10 X B10.BR)F1 B cells were separated into adherent and nonadherent cell populations by their ability to bind to either one of the parental B cell monolayers, and the specificity of inhibition of their responses to B151-TRF2 by anti-Ia antibodies was assessed. It was found that the responses of (B10 X B10.BR)F1 B cells adherent to the B10 B cell monolayer or the B10.BR B cell monolayer were almost completely inhibited by anti-I-Ab and anti-I-Ak antibodies, whereas those of nonadherent cells were now selectively inhibited by anti-I-Ak and anti-I-Ab antibodies, respectively. These findings are interpreted as indicating that the B151-TRF2-responsive F1 B cells consist of at least two subpopulations with the restriction specificity for either one of the parental Ia antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.     

Differential effects of cyclosporin A on diverse B cell activation phenomena triggered by crosslinking of membrane IgM.

Cyclosporin A (CsA) was tested for its modulatory effects on the mIgM-mediated signaling of G0*-associated increases in class II MHC expression, G1-related RNA synthesis, and S phase-related DNA synthesis in human B cells. While CsA at concentrations as low as 10-100 ng/ml could completely ablate anti-IgM-induced DNA synthesis, earlier G1-associated RNA synthesis was only partially inhibited, and signaling of increased membrane class II MHC expression was unaffected by up to 1000 ng/ml of CsA. Similar phenomena were observed in a clonal population of leukemic B lymphocytes susceptible to anti-IgM-mediated activation in the absence of T cells and T cell factors indicating (a) that the inhibitory effects are not due to CsA-mediated suppression of cytokine production by contaminating T cells, and (b) that the varying effects of CsA on the diverse activation phenomena do not reflect B cell subpopulation diversity. Pulsing studies revealed that while maximal suppression of anti-IgM-induced G1-associated RNA synthesis required CsA at culture initiation, near maximal suppression of DNA synthesis occurred when CsA, or soluble human IgM, was added up to 30 hr after the initial exposure of resting B cells to the anti-IgM ligand. These latter findings are consistent with the possibility that the CsA-mediated suppression of S phase entry is due to the inhibition of a signaling event proximal to mIgM ligation which must be repeatedly initiated throughout the first 30 hr of activation.     

Analysis of two distinct B cell activation pathways mediated by a monoclonal T helper cell. I. MHC-restricted activation of B cells by an IL 2-dependent pathway

The present study was carried out to determine whether the MHC-restricted and MHC-unrestricted B cell activation pathways mediated by a single cloned Th cell are separable, and whether these two pathways are mediated by distinct mechanisms. It was demonstrated that the two B cell activating functions of a single cloned Th cell could be separated by their sensitivity to irradiation. It was shown that MHC-restricted B cell activation is mediated by a radiosensitive Th cell function, whereas MHC-unrestricted B cell activation is mediated by a radioresistant function of the same Th cell. In addition, it was shown that recombinant IL 2 can restore or replace the radiosensitive component of MHC-restricted cognate helper function.     

Differential effects of anti-IL-1R accessory protein antibodies on IL-1alpha or IL-1beta-induced production of PGE(2) and IL-6 from 3T3-L1 cells

Soluble or cell-bound IL-1 receptor accessory protein (IL-1RAcP) does not bind IL-1 but rather forms a complex with IL-1 and IL-1 receptor type I (IL-1RI) resulting in signal transduction. Synthetic peptides to various regions in the Ig-like domains of IL-1RAcP were used to produce antibodies and these antibodies were affinity-purified using the respective antigens. An anti-peptide-4 antibody which targets domain III inhibited 70% of IL-1beta-induced productions of IL-6 and PGE(2) from 3T3-L1 cells. Anti-peptide-2 or 3 also inhibited IL-1-induced IL-6 production by 30%. However, anti-peptide-1 which is directed against domain I had no effect. The antibody was more effective against IL-1beta compared to IL-1alpha. IL-1-induced IL-6 production was augmented by coincubation with PGE(2). The COX inhibitor ibuprofen blocked IL-1-induced IL-6 and PGE(2) production. These results confirm that IL-1RAcP is essential for IL-1 signaling and that increased production of IL-6 by IL-1 needs the co-induction of PGE(2). However, the effect of PGE(2) is independent of expressions of IL-1RI and IL-1RAcP. Our data suggest that domain III of IL-1RAcP may be involved in the formation or stabilization of the IL-1RI/IL-1 complex by binding to epitopes on domain III of the IL-1RI created following IL-1 binding to the IL-1RI.     

TNF receptor-associated factor-2 is involved in both IL-1 beta and TNF-alpha signaling cascades leading to NF-kappa B activation and IL-8 expression in human intestinal epithelial cells

Cytokine signaling involves the participation of many adaptor proteins, including the docking protein TNF receptor-associated factor-2 (TRAF-2), which is believed to transmit the TNF-alpha signal through both the I kappa B/NF-kappa B and c-Jun N-terminal kinase (JNK)/stress-related protein kinase (SAPK) pathways. The physiological role of TRAF proteins in cytokine signaling in intestinal epithelial cells (IEC) is unknown. We characterized the effect of a dominant-negative TRAF-2 delivered by an adenoviral vector (Ad5dnTRAF-2) on the cytokine signaling cascade in several IEC and also investigated whether inhibiting the TRAF-2-transmitting signal blocked TNF-alpha-induced NF-kappa B and IL-8 gene expression. A high efficacy and level of Ad5dnTRAF-2 gene transfer were obtained in IEC using a multiplicity of infection of 50. Ad5dnTRAF-2 expression prevented TNF-alpha-induced, but not IL-1 beta-induced, I kappa B alpha degradation and NF-kappa B activation in NIH-3T3 and IEC-6 cells. TNF-alpha-induced JNK activation was also inhibited in Ad5dnTRAF-2-infected HT-29 cells. Induction of IL-8 gene expression by TNF-alpha was partially inhibited in Ad5dnTRAF-2-transfected HT-29, but not in control Ad5LacZ-infected, cells. Surprisingly, IL-1 beta-mediated IL-8 gene expression was also inhibited in HT-29 cells as measured by Northern blot and ELISA. We concluded that TRAF-2 is partially involved in TNF-alpha-mediated signaling through I kappa B/NF-kappa B in IEC. In addition, our data suggest that TRAF-2 is involved in IL-1 beta signaling in HT-29 cells. Manipulation of cytokine signaling pathways represents a new approach for inhibiting proinflammatory gene expression in IEC.     

The role of immunoglobulin receptors and T cell mediators in B lymphocyte activation. I. B cell activation by anti-immunoglobulin and anti-idiotype reagents

The possibility that the failure of anti-mouse immunoglobulin (Ig) antibody to induce antibody synthesis by B cells might be due to reversible receptor blockade was investigated. Murine spleen cells were cultured for 3 days in the presence of minute quantities of intact of (Fab') fragments of rabbit anti-mouse Ig antibody. Thereafter, the cells were washed and either trypsin treated or not before reculturing for 18 hr. Only cells that had been trypsinized after culturing with either intact or fragments of anti-Ig gave a vigorous polyclonal antibody response. This response was extremely T dependent, since T cells or culture supernatants from Con A-activated T cells were required for the B cell response. Moreover, anti-delta was much more effective than anti-mu in inducing antibody synthesis. Finally, the use of three different anti-idiotypic antisera rather than anti-Ig reagents selectively activated the specific idiotype in each instance. The findings demonstrate that anti-Ig reagents can potentiate the response of B cells to signals delivered by T cells.     

Proliferation and production of IL-2 and B cell stimulatory factor 1/IL-4 in early fetal thymocytes by activation through Thy-1 and CD3

To examine which cell surface molecules can operate as transducers of activation signals to early fetal thymocytes, we analyzed the ability of mAb to CD3 and Thy-1 to induce fetal thymocyte activation. Both proliferation and lymphokine secretion were used as measures of activation. We show that anti-CD3 antibodies induce activation of fetal thymocytes as early as day 13 of fetal thymus development, 2 days before CD3 can be detected by flow cytometry. In addition, an alternative activation signal can be delivered to fetal thymocytes through the Thy-1 molecule as early as it is expressed, i.e., day 13. Both CD3- and Thy-1-mediated activation of day 15 fetal thymocytes results in expansion of cells expressing a CD3-gamma delta receptor complex; no CD3-alpha beta receptor complex could be detected. IL-2 production induced by CD3- and Thy-1-induced activation of fetal thymocytes is evident at the 13th day of gestation. Finally, an additional lymphokine B cell stimulatory factor-1 (BSF-1)/IL-4 (so far known only to be produced by mature CD3- cells), is also produced by fetal thymocytes. The results demonstrate that at least two cell surface molecules, Thy-1 and CD3, can function as pathways of activation in fetal thymocytes, and that at least two lymphokines, IL-2 and BSF-1/IL-4, are produced upon activation. These findings may well reflect a role for the early appearance of CD3- cells in thymus ontogeny.     

Differential inhibition of T and B cell function in IL-4-dependent IgE production by cyclosporin A and methylprednisolone

The present study examines the role of the immunosuppressive agents methylprednisolone (MPN) and cyclosporin (Cs)A on IL-4-dependent IgE and IgG production. Addition of optimal amounts of IL-4 (100 U/ml) to cultures of tonsil mononuclear cells resulted in a mean increase in IgG production of 175% and in IgE production of 2460%. Frequency analysis of IgE- and IgG-producing B cells, using an ELISA spot assay, showed parallel increases in both Ig production and numbers of Ig-secreting B cells. IgE production was also enhanced by addition of IL-2 (10 U/ml) and maximal IgE production was obtained with a combination of IL-4 and IL-2. MPN (10(-7) M) and CsA (1 microgram/ml) markedly reduced IL4-induced IgE and IgG production as well as numbers of Ig-secreting cells in a dose-dependent fashion. The suppression of Ig production by the cyclosporins was restricted to the immunosuppressive compounds CsA, CsG, and dihydro-CsD, but not the nonimmunosuppressive drug CsH. Delayed addition of CsA revealed that inhibition was maximal when the drug was added during the first 48 h after addition of IL-4 to the culture. Addition of IL-2 (10 U/ml) partially overcame the inhibition induced by CsA. In coculture experiments, in which separated T or B cells were precultured with the drugs and the cells were then combined and further incubated in the presence of IL-4, the suppressive effects of CsA on IgE production were related to pretreatment of the T but not B cells. The maximum inhibiting effects of MPN were similarly observed when the drug was present in the cultures from the beginning, and addition of IL-2 also partially reversed this inhibition. In contrast to the results with CsA, pretreatment of the B but not T cells with MPN-reduced IgE production. These studies demonstrate that IL-4 increases both numbers of IgE-secreting cells as well as IgE production and CsA and MPN differentially affect the responding T and B cells, resulting in inhibition of Ig production.     

Antigen-specific, MHC-restricted B cell activation by cell-free Th2 cell products. Synergy between antigen-specific helper factors and IL-4

Ag-specific and MHC-restricted Th clones of different Ag specificities and MHC haplotypes were tested for their ability to produce soluble factors capable of providing the signals required for B cell activation and IgG antibody production. Each of five Th clones tested generated significant helper activity in supernatants derived from coculture of the T cell clone with specific Ag and syngeneic APC. The same helper activity was detected in supernatants of clones stimulated with immobilized anti-CD3 antibody in the absence of Ag or APC. The secreted helper activity resembled the activity of the intact Th cells in that it was Ag-specific, carrier-hapten-linked and MHC-restricted. These T cell products functioned to activate only those B cells expressing MHC products which corresponded to the specificity of each Th clone. Thus, the specificity of the cell-free T cell product mimicked precisely that expressed by the intact Th cell and presumably mediated by the cell surface TcR. In addition to the apparent presence of specific helper factor in Th clone supernatants, a role for nonspecific lymphokines was also identified in these preparations. Although recombinant or purified IL-4 alone was not sufficient to stimulate hapten-primed B cells to secrete hapten-specific IgG antibodies, mAb specific for IL-4 blocked the induction of antibody secretion by Th cell supernatant. These results indicate that stimulation of B cells to produce hapten-specific IgG antibody requires at least two distinct signals: an Ag-specific T cell signal which is restricted by MHC products expressed on the B cells, and a nonspecific signal mediated at least in part by the lymphokine IL-4.